Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: Osteoblast adhesion and survival are markedly reduced and Rictor expression is downregulated with aging in vitro and in vivo . ( a ) Representative immunohistochemical (IHC) staining for osteocalcin (OCN) from 3-month-old and 16-month-old mice ( n =6). Arrow, osteoblast; BS, bone surface; scale bar, 50 μ m; * P <0.01 versus 3M. ( b ) SEM image of femora trabecular bones from 3- and 16-month-old mice. Upper panel showed less and thinner trabecula bone in 16-month-old mice. Black arrow, osteoblasts or osteocytes, up panel scale bar, 500 μ m; lower panel scale bar, 50 μ m. ( c ) Photomicrographs of fluorescent calcein staining of 3- and 16-month-old mice and quantification of mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR) and MAR/osteoblast. Arrow: bone formation surface; scale bar, 20 μ m ( n =3 * P <0.01 versus 3M). ( d ) Cell adhesion analyses of calvarial osteoblast cultures from 3- and 16-month-old mice with (lower panel) or without (upper panel) matrigel in the plate ( n =3 * P <0.01 versus 3M). ( e ) Representative photomicrographs of bone nodule formation in MSC cultures from 3- and 16-month-old mice. Scale bar, 400 μ m. ( f ) Western blot analysis of cleaved-poly ADP-ribose polymerase (PARP) in lysates of trabecular bone dissected from femora of 3- and 16-month-old mice. ( g ) Western blot analysis of Rictor and P-Akt(S473) expression in lysates of trabecular bone from 3- and 16-month-old mice. ( h ) Western blot analysis of mTOR, Rictor, Raptor and P-Akt(S473) expression in lysates of lung, spleen and kidney from 3- and 16-month-old mice. ( i ) Western blot analysis of Rictor, mTOR, Raptor, osteocalcin (OCN) and P-Akt(S473) expression in primary calvarial osteoblasts from 3- and 16-month-old mice

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Expressing, In Vitro, In Vivo, Immunohistochemical staining, Immunohistochemistry, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: Rictor is a target of miR-218 in osteoblasts. ( a ) Real-time PCR analysis of 13 miRNAs expression in trabecular bone from 3- and 16-month-old mice ( n =6). * P <0.01 versus 3M. ( b ) Western blot analysis of Rictor, p-Paxillin(Y118) and P-Akt(S473) expression in osteoblasts transfected with miR-218 mimics and miR-218 inhibitors. ( c ) The putative miR-218 binding site in the Rictor 3′ UTR. ( d ) Luciferase activity in MC3T3-E1 cells co-transfected with miR-218 mimics or negative control (NC) and the indicated 3′ UTR-driven reporter constructs ( n =3). * P <0.01 versus NC; ** P <0.05 versus NC

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Binding Assay, Luciferase, Activity Assay, Negative Control, Construct

Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: miR-218 represses osteoblast adhesion and survival by targeting Rictor. ( a ) Adhesion analysis of MC3T3-E1 cells transfected with miR-218 mimics, miR-218 inhibitors and NCs ( n =3). * P <0.01, ** P <0.05. ( b ) AnnexinV analysis of apoptosis in MC3T3-E1 cells transfected with miR-218 mimics or miR-218 inhibitor and serum-starved for 48 h ( n =3). * P <0.01, ** P <0.05. ( c ) Western blot analysis of Rictor, p-Paxillin(Y118), Runx2 and p-Akt(S473) expression in MC3T3-E1 cells transfected with miR-218 mimics. ( d ) Western blot analysis of Rictor, p-Paxillin(Y118), Runx2 and p-Akt(S473) expression in MC3T3-E1 cells transfected with miR-218 inhibitor or NCs. ( e ) Western blot analysis of Rictor, p-Paxillin(Y118) and p-Akt(S473) expression in MC3T3-E1 cells transfected with miR-218 mimics and/or Rictor-expressing vectors. ( f ) Adhesion analysis of MC3T3-E1 cells transfected with miR-218 mimics and/or Rictor-expressing vector ( n =3). * P <0.01, ** P <0.05. ( g ) AnnexinV analysis of apoptosis in MC3T3-E1 cells transfected with miR-218 mimics and/or Rictor-expressing vector ( n =3); * P <0.01, ** P <0.05

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: ROS stimulates miR-218 to downregulate Rictor in aged mice osteoblast. ( a ) ROS levels in trabecular bones of 3- and 16-month-old mice ( n =6); * P <0.01 compared with 3-month-old mice. ( b ) Effect of H 2 O 2 (1 μ M) on miR-218 expression in MC3T3-E1 cells ( n =3). * P <0.01 versus controls. ( c ) Western blot analysis of Rictor, mTOR and Raptor expression in MC3T3-E1 cells treated with H 2 O 2 (5, 10 μ M) for 48 h. ( d ) Effect of H 2 O 2 (1 μ M for 24 h) on MC3T3-E1 cell adhesion ( n =3). * P <0.01 versus controls. ( e ) Effect of H 2 O 2 (10 μ M for 36 h) and on MC3T3-E1 cell apoptosis( n =3). * P <0.01 versus controls

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Loss of Rictor with aging in osteoblasts promotes age-related bone loss

doi: 10.1038/cddis.2016.249

Figure Lengend Snippet: ROS scavenger reduces miR-218 and Rictor expression and aged-related bone loss in mice. ( a ) Western blot analysis of Rictor, Raptor and P-Akt(S473) expression in trabecular bone of 3-, 9- and 16-month-old mice. ( b ) Trabecular bone ROS levels in 9-month-old mice treated with N-acetyl- l -cysteine (NAC; 2 mg/ml) or vehicle for 7 months ( n =10). * P <0.01 versus controls. ( c ) Representative micro-CT scans of vertebrae from mice described in ( b ). ( d – g ) Trabecular bone BV/TV, BMD, Tb.Sp and Tb.N in mice described in ( c ) ( n =10); * P <0.01 versus controls. ( h , i ) Oestocalcin IHC ( h ) or TRAP ( i ) staining of distal femora from mice described in ( b ). Scale bar, 50 μ m; * P <0.01 versus controls. ( j ) Real-time PCR analysis of miR-218 expression in trabecular bones of mice described in ( b )( n =10). * P <0.01 versus controls. ( k ) Westem bolt analysis of c-PARP expression in trabecular bones of mice described in (b). ( l ) Western blot analysis of Rictor, Raptor and P-Akt(S473) expression in trabecular bones of mice described in ( b ). ( m ) A schematic model depicting that the upregulation of miR-218 and loss of Rictor with aging induces the pathogenesis of age-related bone loss

Article Snippet: Cells were transfected with a Rictor expression vector (pRK5-myc-Rictor, addgene, cat #1860), or with 100 nM miR-218 mimic, miR-218 inhibitor or scramble Control oligo using the transfection agent lipo2000 (Invitrogen, Carlsbad, Canada) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Micro-CT, Staining, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

doi: 10.1186/bcr3611

Figure Lengend Snippet: Runx2 specifically maintains pAkt in invasive mammary epithelial cells. A) The basal levels of pAkt (Serine 473) expression was examined in MDA-MB-231, SUM-159 and SUM-159-PT cells cultured in regular growth medium by Western blotting. B) The Runx2 protein expression was stably suppressed in SUM-159 cells by lentivirus-mediated Runx2 shRNA delivery. The Runx2 expression levels were analyzed by Western blotting while β-Actin was used as the loading control. C) The SUM-159 cells expressing Runx2-RNAi or control were serum-deprived and stimulated with 100 ng/ml of EGF in the presence of 20 μM LY294002. The whole cell lysates were analyzed for pAkt (Serine 473) and total Akt expression by Western blotting at 10 minutes post epidermal growth factor (EGF) stimulation. A quantification of normalized relative pAkt levels is given below respective lane. D) The SUM-159-PT cells were transfected with dsRNA targeting a different Runx2 mRNA sequence to transiently suppress Runx2 expression. E) The serum-deprived, EGF stimulated SUM-159-PT cells were analyzed for pAkt (Serine 473) and Akt levels by Western blotting. A quantification of normalized pAkt level is shown below the blot. F) The MDA-MB-231 cells expressing Runx2-RNAi or control were serum-deprived and stimulated with 100 ng/ml of EGF in the presence of 20 μM LY294002. The whole cell lysates were analyzed for pAkt (Serine 473) and total Akt expression by Western blotting at 10 minutes and 1 hour post EGF stimulation.

Article Snippet: The lentivirus vector expressing Rictor shRNA was obtained from Addgene (plasmid #1853) (Cambridge, MA, USA) [ ].

Techniques: Expressing, Cell Culture, Western Blot, Stable Transfection, shRNA, Control, Transfection, Sequencing

Journal: Breast Cancer Research : BCR

Article Title: Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

doi: 10.1186/bcr3611

Figure Lengend Snippet: Reducing PHLPP1 phosphatase and pERK kinase activity rescues Runx2-mediated inhibition of pAkt in MDA-MB-231 cells. A) The PHLPP1 mRNA expression was transiently suppressed in MDA-MB-231 cells and gene expression was analyzed by real-time PCR. A quantification of GAPDH normalized PHLPP1 mRNA expression is shown. B) A quantification of the relative phosphorylation levels of pAkt (Serine 473) were determined by Western blotting in PHLPP1 knockdown MDA-MB-231 cells. C) The levels of pAkt (Serine 473) and total Akt proteins were analyzed by Western blot in PHLPP1 knock out control or Runx2 suppressed MDA-MB-231 cells as indicated. A quantification of pAkt levels normalized to total Akt protein is shown below the respective lane. D) The control or Runx2 knockdown MDA-MB-231 cells were stimulated with EGF for indicated times in the presence or absence of ERK inhibitor U0126. The level of pAkt and Akt was determined by Western blotting and quantification is shown below the respective lane. E) The Runx2 gene expression was conditionally suppressed by doxycycline in MDA-MB-231 cells stably expressing tTR-KRAB and Runx2-shRNA. The serum-deprived cells were stimulated with EGF in the presence of ERK inhibitor PD184161 for indicated times. The pAkt and total Akt expression was analyzed by Western blotting and a quantification of normalized expression is shown below the respective lanes.

Article Snippet: The lentivirus vector expressing Rictor shRNA was obtained from Addgene (plasmid #1853) (Cambridge, MA, USA) [ ].

Techniques: Activity Assay, Inhibition, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Knockdown, Knock-Out, Control, Stable Transfection, shRNA

Journal: Breast Cancer Research : BCR

Article Title: Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

doi: 10.1186/bcr3611

Figure Lengend Snippet: Runx2 knockdown alters expression levels of mTORC-2 proteins. A) The MDA-MB-231 cells’ transient Runx2 suppression was analyzed for mTOR and Runx2 levels. B) A quantification of mTOR protein expression normalized to β-Actin is shown. C) The stable Runx2 knockdown MDA-MB-231 cells were serum-deprived, epidermal growth factor (EGF) stimulated and examined for pmTOR (Serine 2481) and mTOR (total) protein. D) The Runx2 knockdown cells were assayed for mTOR gene expression by RT-PCR (normalized to GAPDH ). E) The Runx2 knockdown and Ad-GFP- or WT-Runx2-treated MDA-MB-231 cells were tested for Runx2 recruitment on mTOR promoter by ChIP assays. A schematic diagram of the mTOR promoter region is bars indicating potential Runx binding sites (black bars) and location of PCR primers (small arrows). F) and G) The mRNA (F) and protein (G) expression levels of Rictor were analyzed in control or Runx2 knockdown MDA-MB-231 cells by RT-PCR and Western blotting, respectively. H) The MDA-MB-231 cells stably over-expressing WT-Runx2 were assayed for Runx2 and Rictor gene expression by RT-PCR. I) The MDA-MB-231 cells with stable Rictor knockdown were stimulated with EGF and were analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor protein levels by Western blotting. J) The stably Runx2 knockdown MDA-MB-231 cells together with Rictor suppression were stimulated with EGF and analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor proteins by Western blotting. K) The glucose and serum-deprived (24 h) MDA-MB-231 cells with Runx2 knockdown (Runx2-RNAi) together with Rictor knockdown (Rictor -shRNA) were stained for Annexin V and AAD by flow cytometry. L) A quantification of positive cells with or without Rictor knockdown is shown. M) A schematic diagram depicting the function of Runx2 in regulating Akt via mTORC2 complex and its effect on survival of invasive cancer cells.

Article Snippet: The lentivirus vector expressing Rictor shRNA was obtained from Addgene (plasmid #1853) (Cambridge, MA, USA) [ ].

Techniques: Knockdown, Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Control, Western Blot, Stable Transfection, shRNA, Staining, Flow Cytometry